Recombinant DNA sequences, vectors containing them and method for the use thereof

Recombinant DNA sequences which encode the complete amino acid sequence of a glutamine synthetase, vectors containing such sequences, and methods for their use, in particular as dominant selectable markers, for use in co-amplification of non-selected genes and in transforming host cell lines to glutamine independence are disclosed.

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Claims

1. A recombinant DNA expression vector which is amplifiable in a transformed host cell and which encodes the complete amino acid sequence of a mammalian glutamine synthetase (GS).

2. A host cell transformed with a vector according to claim 1.

3. A recombinant DNA expression vector according to claim 1, wherein the mammalian glutamine synthetase (GS) has the amino acid sequence of the chinese hamster GS of FIGS. 2a to 2d.

4. A vector comprising a gene which encodes the complete amino acid sequence of a glutamine synthetase (GS) and a gene which encodes the amino acid sequence of a desired protein other than said GS.

5. A method of co-amplifying a recombinant DNA sequence which encodes the complete amino acid sequence of a desired protein other than a glutamine synthetase (GS), comprising:

(a) transforming a eukaryotic host cell with the expression vector of claim 4; and
(b) culturing said transformed host cell under conditions which allow transformants containing an amplified number of copies of the vector to be selected.

6. Plasmid pSVLGS.tPA16.

7. Plasmid pSVLGS.tPA17.

8. An expression vector for co-amplifying a recombinant DNA which encodes the amino acid sequence of a desired protein other than a glutamine synthetase (GS) comprising:

(a) a recombinant DNA which encodes the complete amino acid sequence of a GS; and
(b) a recombinant DNA which encodes the complete amino acid sequence of a desired protein other than said GS, wherein the GS and desired protein coding DNAs are linked such that amplification of the GS coding sequence results in co-amplification of the desired protein coding DNA.

9. An amplifiable recombinant DNA which encodes a complete, enzymatically active glutamine synthetase (GS).

10. A host cell transformed by a recombinant DNA expression vector according to claim 3.

11. The method of claim 5, wherein step (b) comprises culturing the transformed host cell in media containing a GS inhibitor and selecting for transformed cells which are resistant to progressively increased levels of the GS inhibitor.

12. The method of claim 11, wherein the GS inhibitor is selected from the group consisting of phosphinothricin and methionine sulphoxime.

Referenced Cited
U.S. Patent Documents
4399216 August 16, 1983 Axel et al.
4656134 April 7, 1987 Ringold
4797359 January 10, 1989 Finkelstein
5122464 June 16, 1992 Wilson et al.
Foreign Patent Documents
WO8606409 April 1986 WOX
Other references
  • Alberts et al. "Molecular Biology of the Cell", Garland Publishing Inc., New York, 1983, pp. 184-193. Watson et al. "Recombinant DNA, A Short Course" W.H. Freeman and Co., 1983, pp. 50-90. Sanders, Peter G. et al., "Amplification and cloning of the Chinese hamster glutamine synthetase Gene", EMBO, vol. 3, No. 1, pp. 65-71 (1984). Pennica, Diane et al., "Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli", Nature, vol. 301, pp. 214-221 (1983). Donn, Gunter et al., "Herbicide-Resistant Alfalfa Cells: An Example of Gene Amplification in Plants", Journal of Molecular and Applied Genetics, 2:621-635 (1984). Young, Anthony P. et al., "Mouse 3T6 Cells That Overproduce Glutamine Synthetase", The Journal of Biological Chemistry, vol. 258, No. 18, pp. 11260-11266 (1983). de Saint Vincent, Bruno Robert et al., "The Cloning and Reintroduction into Animal Cells of a Functional CAD Gene, a Dominant Amplifiable Genetic Marker" Cell, vol. 27, pp. 267-277 (1981). Murray, Mark J. et al., "Construction and Use of a Dominant . . . ", Molec. and Cell Biol., vol. 3, No. 1, pp. 32-43 (1983). Kaufman, Randal J., et al., "Selection and Amplification of Heterologous Genes . . . ", Proc.Natl. Acad.Sci. U.S.A., vol. 83, pp. 3136-3140 (1986). Kabak, David B. et al., "Ribosomal DNA Magnification in Saccharomyces cerevisiae", Bacteriol, vol. 134, pp. 237-245 (1978). Vel'Kov, V.V., "Amplification of Genes in Prokaryotic and Eukaryotic Systems", Soviet Genetics, vol. 18, pp. 348-396 (1982).
Patent History
Patent number: 5770359
Type: Grant
Filed: Sep 8, 1994
Date of Patent: Jun 23, 1998
Assignees: Celltech Therapeutics Limited (Berkshire), The University Court of the University of Glasgow (Glasgow)
Inventors: Richard Harris Wilson (Glasgow), Christopher Robert Bebbington (Windsor)
Primary Examiner: David Guzo
Law Firm: Spencer & Frank
Application Number: 8/302,241